--- - |2- Abstract Human mesenchymal stem cells (hMSCs) are fibroblastoid multipotent adult stem cells with capacities of differentiation into osteoblasts and chondrocytes and show great potential in new bone formation and bone repair‐related clinical settings, such as osteoporosis. Long noncoding RNAs (lncRNAs) have been demonstrated to play important roles in various biological processes. Here, we report an antisense lncRNA SEMA3B‐AS1 regulating hMSCs osteogenesis. SEMA3B‐AS1 is proximal to a member of the semaphorin family Sema3b. Overexpression of SEMA3B‐AS1 using the lentivirus system markedly inhibits the proliferation of hMSCs and meanwhile reduces osteogenic differentiation. Using a comprehensive proteomic technique named isobaric tag for relative and absolute quantitation, we found that SEMA3B‐AS1 significantly alters the process of osteogenesis through downregulating the expression of proteins involved in actin cytoskeleton, focal adhesion, and extracellular matrix–receptor interaction, while increasing the expression of proteins in the spliceosome. Collectively, we find that SEMA3B‐AS1 is a target for controlling osteogenesis of hMSCs. - Journal of Cellular Physiology, EarlyView.